Descendants of bacteria devoid of N terminal formylation useful for the production of proteins and peptides

ABSTRACT

The present invention relates to producing non-formylated proteins and/or peptides in bacterial cells lacking deformylase and/or transformylase.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/303,065, which was filed on Jul. 6, 2001.

FIELD OF THE INVENTION

[0002] The present invention is useful for production of recombinantproteins in eubacterial hosts. A eubacterial host defective in the genesfor Met-tRNAi transformylase and polypeptide deformylase is describedwhich grows in minimal and complex nutrient media at 30° C., 37° C. and42° C. with near wild-type rate. In this eubacterium protein synthesisdoes not require N-formyl methionine as the initiator methionine,protein synthesis instead is initiated with unmodified methionine. Theabsence of peptides which retain N-formyl methionine in this eubacteriummakes it particularly suited for the expression of recombinant proteinsfor pharmaceutical use

BACKGROUND OF THE INVENTION

[0003] In eubacteria peptide synthesis is initiated at methionine startcodons which are read by N-formyl methionine tRNA. Prior to translationinitiation the methionyl moiety of the charged tRNA is N-formylated bythe action of Met-tRNAi transformylase (E.C.2.1.2.9). The N-formyl groupis removed from the native protein by polypeptide deformylase (E.C.3.5.127), and the initiator methionine can then be cleaved off bymethionine aminopeptidase, completing the primer methionine cycle. Incontrast, archaea and eukaryotes have a primer methionine cycle devoidof N-formylating and deformylating activities (for review see Mazel, D.,Pochet, S. and Marliere, P.: Genetic characterization of polypeptidedeformylase, a distinctive enzyme of eubacterial translation. EMBO J. 13(1994) 914-923, and Mazel et al 1996).

[0004] Expression of eukaryotic proteins in eubacterial hosts oftenresults in the production of recombinant proteins that retain anN-terminal formylmethionyl residue (examples include bovine somatotropin(Bogosian, G., et al., (1989) Biosynthesis and incorporation intoprotein of norleucine by Escherichia coli J. Biol Chem. 264:531-539.);eel growth hormone (Sugimoto, S., Yamaguchi, K. and Yokoo, Y.: Isolationand characterization of recombinant eel growth hormone expressed inEscherichia coli. J. Chromatog. 515 (1990) 483-494); human granulocytecolony-stimulating factor (Clogston, C. L., Hsu, Y. R, Boone, T. C. andLu, H. S.: Detection and quantitation of recombinant granulocytecolony-stimulating factor charge isoforms: comparative analysis bycationic-exchange chromatography, isoelectric focusing gelelectrophoresis, and peptide mapping. Anal. Biochem. 202 (1992)375-383.); bovine fatty acid-binding protein (Specht, B.,Oudenampsen-Kruger, E., Ingendoh, A., Hillenkamp, F., Lezius, A. G. andSpener, F.: N-terminal variants of fatty acid-binding protein frombovine heart overexpressed in Escherichia coli. J. Biotechnol. 33 (1994)259-269); bovine cytochrome P450 (Dong, et al FASEB J. 9 (1995) A1486);Methanothermus fervidus histone A (Sandman, K., Grayling, R. A., andReeve, J. N. Improved N-terminal Processing of Recombinant ProteinsSynthesized in Escherichia coli. Biotechnology 13 (1995) 504-506); humaninterleukin-5 (Rose, K., Regamey, P., Anderegg, R, Wells, T., Proudfoot,A., Human interleukin-5 expressed in Escherichia coli has N-terminalmodifications. Biochem J. 286 (1992) 825-828); human parathyroid hormone(Rabbani, S. A., Yasuda T., Bennett H. P. J., Sung, W. L. Zahab, D. M.,Tam, C. S. Goltman, D., and Hendy, G. N. Recombinant Human ParathyroidHormone Synthesized in Escherichia coli. Journal of Biological Chemistry263:3 (1988) 1307-1313; Hogset, A., Blingsmo, O. R., Gaurvk V. T.,Saether, O., Jacobsen, P. B., Gordeladzo, J. O., Alestrom, P. andGautvik, K. M. Expression of Human Parathyroid Hormone In Escherichiacoli. Biochemical and Biophysical Research Communications 166:1 (1990)50-60); human gamma-interferon (Honda, S., Asano, T., Kajio, T., andNishimura, O. Escherichia coli—Derived Human Interferon—.gamma. withCys-Tyr-Cys at the N-Terminus is Partially Nα-Acylated. Archives ofBiochemistry and Biophysics 269 (1989) 612-622)). In addition retentionof N-formyl methionine has been found in endogeneous E. coli proteins(Hauschild-Rogat, P. N-formylmethionine as a N-terminal group of E. coliribosomal protein. Mol. Geri. Genet. 102 (1968) 95-101, Marasco, W. A.,Phan, S. H., Kruusch, H., Showell, H. J., Feltner, D. E., Naim, R,Becker, E. L. and Ward, P. A.: Purification and identification offormyl-methionyl-leucyl-phenylalanine as the major peptide neutrophilchemotaetic factor produced by Escheriehia coli. J. Biol. Chem. 259(1984) 5430-5439; Milligan, D. L. and Koshland, Jr., D. E.: The aminoterminus of the aspartate chemoreceptor is formylmethionine. J. Biol.Chem. 265 (1990) 4455-4460). Since N-formylated peptides are a majorindicator of eubacterial infections for the mammalian immune system andare highly immunogenic, incomplete deformylation precludes; for example,the use of N-formylated preparations for therapeutic purposes. Severalapproaches to circumvent this problem have been proposed, e.g.,expression in the presence of trimethoprim and thynidine (Sandman et aL,1995), overexpression of peptide deformylase in the host (Warren, W. C.,Bentle, K. A., Schlittler, M. R, Schwane, A. C., O'Neil, J. P. andBogosian, G. (1996): increased production of peptide deformylaseeliminates retention of formylmethionine in bovine somatotropinoverproduced in Escherichia coli. Gene 174, 235-23), expression as aprotein fusion either with an N-terminal peptide that can be removed invitro by a specific protease or with an N-terminal leader peptide whichis cleaved during transport of the nascent protein in anon-cytoplasmatic compartment. Finally, the N-formyl group may also beremoved by mild acid hydrolysis, or the fraction of the proteinretaining N-formyl methionine may be separated from the correctlyprocessed protein by purification procedures.

[0005] Each of these approaches has significant disadvantages. Additionof trimethoprim and thymidine is costly, requires manipulation of theculture that will express the recombinant protein, and may slow downgrowth of the host. Overexpression of peptide deformylase requires astable plasmid construct in the host that has to be selected for;moreover, deformylation may be less than 100% effective. Expression offusion proteins requires exact molecular constructions; chemicalhydrolysis with acid may cause dammage to the rest of the protein.Finally, none of these approaches guarantees a final preparation that isabsolutely free of N-formylated peptides derived either from therecombinant protein or from contaminations with endogeneous hostpeptides.

SUMMARY OF THE INVENTION

[0006] The present invention is useful for production of recombinantproteins in eubacterial hosts. A eubacterial host defective in the genesfor Met-tRNAi transformylase and polypeptide deformylase is describedwhich grows in minimal and complex nutrient media at 30° C., 37° C. and42° C. with near wild-type rate. In this eubacterium protein synthesisdoes not require N-formyl methionine as the protein initiator proteinsynthesis instead being initiated with unmodified methionine. Theabsence of peptides which retain N-formyl methionine in this eubacteriummakes it particularly suited for the expression of recombinant proteinsfor pharmaceutical use.

[0007] The present invention relates to the use of a bacterial strainlacking of transformylase and deformylase activity for the production ofnon formylated peptides or proteins and also the selection of a mutantbacteria which is not spntaneousely reversible to the wild type for aformylase and deformylase activities but has a doubling generation timesimilar to the growth rate of the wild type bacteria.

[0008] More particularly the invention provides the use of a bacterialstrain lacking of deformylase and/or transformylase activitiescharacterized in that it is non reversible for the deformylase and/ortransformylase activities

[0009] The invention also provides the use of the bacterial strainaccording to the invention characterized in that it developes at atemperature of 37° C.

[0010] The invention further relates the use of a bacterial strainaccording to the invention characterized in that the bacteria is E. coli

[0011] The invention also provides bacteria strain lacking of theprotein deformylation and/or trans formylation activity characterized inthat

[0012] said bacteria is not able to revert spontaneousely for theseactivities,

[0013] the growth rate of said bacteria is at least equivalent to thewild type growth rate.

[0014] The invention is also concerned with the use of the bacteriaaccording to the invention for production of non formylated homologousor heterologous peptides or proteins.

[0015] The invention also relates to a preparation process of peptidesor proteins comprising the following steps:

[0016] a)culture of the bacteria according to the invention

[0017] b) transforming the bacteria according to step a) with a plasmidor a vector comprising an insert containing a polynucleotide coding foran homologous or heterologous peptide or protein

[0018] c) production of the peptide or the protein by the bacteria

[0019] d) optionally, separation of the peptide or protein of interestof the bacterial culture.

[0020] Another object of the invention concerns a process according tothe invention in which the bacteria comprises a polynucleotide codingfor a mutator allele and a process according the invention in which thebacteria mutator phenotype has been complemented or the mutator allelehas been replaced by the wide type allele.

[0021] The invention also concerned a purified polynucleotide comprisinga gene coding for a mutator allele and contained in the bacteria strainaccording to the invention and a purified polynucleotide according tothe invention, in which the mutator phenotype has been complemented orthe mutator allele has been replaced by the wild type allele

[0022] The invention further relates to an E. coli bacterial 2045culture deposited at the CNCM on Jul. 5, 2001 (accession number: 1-2694)

[0023] Another aspect of the invention is directed to a process for theselection of mutants of a bacteria according to the inventioncharacterized by the culture under continuous proliferation conditionsof said mutants and separation of the mutant of interest from the staticbacteria.

[0024] The invention is also concerned with a purified protein obtainedafter the expression of the heterologous or homologous insert ofinterest and deprived of formyl residue.

[0025] Other objects and advantages of the present invention will beapparent upon reading the following non restrictive detailed descriptionmade with reference to the accompanying drawing,

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1: The primer methionine cycle in eubacteria (1 a) andarchaea and eukaryotes (1 b). metG, met-tRNA synthetase; fmt, met-tRNAitransformylase; def, polypeptide deformylase; map, methionineaminopeptidase; aa, amino acid; f, formyl; pp, polypeptide. Modifiedafter (Mazel, D., Pochet, S. and Marliëre, P. (1994): Geneticcharacterization of polypeptide deformylase, a distinctive enzyme ofeubacterial translation. EMBO J. 13, 914-923).

[0027]FIG. 2: In vivo evolution of a D(def-fmt) mutant under permanentproliferation. (2 a) Cells were kept under permanent proliferation inminimal medium at 37° C. A turbidostat regime at 5×108 cells/ml wasapplied. Growth rates are averaged over 24 h periods. Two independentruns are shown. (2 b) Input (1) and evolved strains isolated during theprocess (2, 3, 4; c.f., numbers and open circles in a) were grown onminimal agar for 36 h at 37° C.

[0028]FIG. 3: In vivo evolution of a D(def-f mt) mutant at increasedmutation rates. Two independent runs are shown.

[0029]FIG. 4: Emergence and selection of adhesive variants in aconventional turbidostat, and counter-selection of adhesive variants byEvologic's process. Starting at point 1, adhesive variants were allowedto compete with cells in suspension, periodic destruction of staticvariants was re-established at point 3. (4 a) Growth rates of thepopulations as measured in batch culture. (4 b) Adhesion of cellmaterial to glass surfaces. Isolates (points 1-6 in a) were cultivatedin glass tubes for 20 h at 37° C. Arrowheads point to material thataccumulated on the surface during cultivation.

DETAILED DESCRIPTION OF THE INVENTION

[0030] PEPTIDE: includes any natural or synthetic compounds containingtwo or more amino acids. Therefore, it comprises proteins,glycoproteins, and protein fragments derived from pathogenic organismssuch as viruses, bacteria, parasites and the like, or proteins isolatedfrom normal or pathogenic tissues, such as cancerous cells. It alsoincludes proteins and fragments thereof produced by recombinant meansthat has been associated or not with other peptides coding for tumoral,viral, bacterial or fungic epitopes for forming a fusion. protein

[0031] POLYNUCLEOTIDE: any DNA, RNA sequence or molecule having onenucleotide or more, including nucleotide sequences encoding a completegene. The term is intended to encompass all nucleic acids whetheroccuring naturally or non naturally in a particular cell, tissue ororganism. This includes DNA and fragments thereof, RNA and fragmentsthereof, cDNAs and fragments thereof, expressed sequence tags,artificial sequences including randomized artificial sequences.

[0032] VECTOR: a self replicating RNA or DNA molecule which can be usedto transfer an RNA or DNA segment from one organism to another. Vectorsare particulaly useful for manipulating genetics constructs anddifferents vectors may have properties particularly appropriate toexpress proteins in a recipient during cloning procedures and maycomprise different selectable markers, Bacterial plasmids are commonlyused vectors.

[0033] The present invention describes an E. coli strain with a primermethionine cycle similar to that in eucatyotic cells. This strain nolonger harbors the def fmt operon encoding Met-tRNAi transformylase andpolypeptide deformylase and thus can not N-formylate Met-tRNAi. Removalof N-formyl groups from expressed proteins by any of the techniquesdescribed above is thus no longer required.

[0034] The fmt and def genes from E. coli were previously isolated(Guillon, J. M., Mechulam, Y., Schmitter, J. M., Blanquet, S. and Fayat,G.: Disruption of the gene for Met-tRNA-Net formyltransferase severelyimpairs growth of Escherichia coli. J. Bacteriol. 174 (1992) 4294-4301;Mazel, D., Pochet, S. and Marliere, P.: Genetic characterization ofpolypeptide deformylase, a distinctive enzyme of eubacterialtranslation. EMBO J. 13 (1994) 914-923) (def amino acid sequence isshown in SEQ ID NO: 1, def nucleotide sequence is shown in SEQ ID NO:2;fmt amino acid sequence is shown in SEQ ID NO:3, and fmt nucleotidesequence is shown in SEQ ID NO:4) and shown to be highly conserved amongeubacteria (Mazel, D., Pochet, S. and Marliere, P.: Geneticcharacterization of polypeptide deformylase, a distinctive enzyme ofeubacterial translation. EMBO J. 13 (1994) 914-923 ). Deletion mutantsfor either the fmt gene (Guillon, J. M., Mechulam, Y., Schmitter, J. M.,Blanquet, S. and Fayat, G.: Disruption of the gene for Met-tRNA-Netformyltransferase severely impairs growth of Escherichia coli. J.Bacteriol. 174 (1992) 4294-4301) or the entire def-fit operon (Mazel,D., Pochet, S. and Marliere, P.: Genetic characterization of polypeptidedeformylase, a distinctive enzyme of eubacterial translation. EMBO J. 13(1994) 914-923, D[def-fmt]) were created. The resulting mutants werereported to be severely impaired in growth. The fmt mutant has an8.61-fold decreased growth rate at 37° C. in rich medium and does notgrow at 42° C. (Guillon, J. M., Mechulam, Y., Schmitter, J. M.,Blanquet, S. and Fayat, G.: Disruption of the gene for Met-tRNA-Netformyltransferase severely impairs growth of Escherichia coli. J.Bacteriol. 174 (1992) 4294-4301). The def-fmt mutant has a similarlydecreased growth rate in minimal medium at 37° C., and growth iscompletely impaired in this medium at 42° C. (Mazel, D., Pochet, S. andMarliere, P.: Genetic characterization of polypeptide deformylase, adistinctive enzyme of eubacterial translation. EMBO J. 13 (1994)914-923). Whereas deletion of the fmt gene alone leaves the mutantbacteria viable, deletion of the def gene alone as well asre-introduction of the fmt gene into a def-fmt background is lethal(Mazel, D., Pochet, S. and Marliere, P.: Genetic characterization ofpolypeptide deformylase, a distinctive enzyme of eubacterialtranslation. EMBO J. 13 (1994) 914-923), demonstrating that essentialbacterial proteins either have to be deformylated, and/or that theinitiator methionine has to be cleaved off in order to render theseproteins functional.

[0035] For the purpose of the present invention, a def-fin deletionmutant was selected for enhanced growth rates under permanentproliferation in minimal medium at 37° C. until its growth rateapproximated that of the parent wild-type bacterium. For a secondapplication; the def-fmt deletion mutant previously selected for growthat 37° C. was selected for enhanced growth rates under permanentproliferation in minimal medium at 42° C. until its growth rateapproximated that of the parent wild-type bacterium.

[0036] eubacterium with altered translational mechanism such that itcontains no fmt and def genes yet grows at wt rate.

[0037]E. coli formyl-free strain growing at temperatures higher than 37°C.

[0038] use of such strains for expression of recombinant proteins.

[0039] use of such strains for production of any product in E. coliwhich must not be contaminated with N-formylated peptides

EXAMPLES

[0040] 1. Selection of enhanced growth rate in the def-fmt mutant FIG.2. Growth rate of evolved strains at 30° C., 37° C. and 42° C. inminimal medium and rich medium (table)

[0041] An E. coli strain for expression of N-formyl-free polypeptides toeubacteria peptide synthesis is initiated at methionine start codonwhich are read by N-formyl methionine tRNA. Prior to translationinitiation the methionyl moiety of the charged tRNA is N-formylated bythe action of Met-tRNAi transformylase. The N-formyl group is removedfrom the native protein by polypeptide deformylase, and the initiatormethionine can then be cleaved off by methionine aminopeptidase,completing the primer methionine cycle (FIG. 1a). In contrast, archaeaand eukaryotes have a primer methionine cycle devoid of N-formylatingand deformylating activities (FIG. 1b).

[0042] Expression of eukaryotic proteins in eubacterial hosts oftenresults in the production of recombinant proteins that retain anN-terminal formylmethionyl residue. Since N-formylated peptides arehighly immunogenic, incomplete deformylation precludes, for example,their use for therapeutic purposes. Several approaches to circumventthis problem have been proposed, e.g., expression in the presence oftrimethoprim and thymidine (Sandman, K., Gryling. R. A. and Reeve, J. N.(1995): Improved N-terminal processing of recombinant proteinssynthesized in Escherichia coli. Biotechnology 13, 504-506) oroverexpression of peptide deformylase in the host (Warren, W. C.,Bentle, K. A., Schlittler, M. R, Schwane, A. C., O'Neil, J. P. andBogosian, G. (1996): increased production of peptide deformylaseeliminates retention of formylmethionine in bovine somatotropinoverproduced in Escherichia coli. Gene 174, 235-238).

[0043] The inventors have opted for a radical solution, simplifying theprimer methionine cycle in Escherichia coli by deletion of the def-fmtoperon that encodes polypeptide deformylase and met-tRNAitransformylase, and improving the resulting, crippled strain, byselecting for increasing growth rates (and therefore improved rates ofprotein synthesis) under permanent proliferation in suspension. Theinventors have isolated the def and fmt genes from E. coli and created adeletion mutant (D[def-fmt]j) devoid of both genes (Mazel, D., Pochet,S. and Marliëre, P. (1994): Genetic characterization of polypeptidedeformylase, a distinctive enzyme of eubacterial translation. EMBO J.13, 914-923). The resulting strain was found to be viable, however itsgrowth rate was dramatically reduced, from 0.9 per h to 0.25 per h inminimal medium at 37° C.

[0044] Protein synthesis in living cells is dependent on the concertedaction of a complex assembly of the protein and rRNA constituents ofribosomes and a host of factors catalyzing aminoacylation of tRNAs,initiation, elongation and termination of translation as well asmaturation of nascent polypeptides. N-terminal formylation is among themost conserved features that distinguish eubacteria from archaea andeukaryotes. Removing the enzymes that catalyze the correspondingreactions is therefore expected to remove the efficiency of proteinsynthesis far from its wild-type optimum. Evolutionary ressurection fromthis type of genetic injury will require multiple adaptive mutations torender the bacterial translation machinery more similar to that found ineukaryotes. State-of-the-art technologies for directed evolution ex vivoare unable to predict and select the adaptive mutations that wouldre-establish wild-type protein synthesis rates in a D(def-fmt)background.

[0045] In vivo evolution of the D(def=fmt) mutant under permanentproliferation in suspension in a turbidostat regime yields variants withincreasing growth rate; approximating wild-type growth rate after about1 month (ca. 300 generations) of permanent selection (FIG. 2a). FIG. 2bshows drastically increased biomass production of evolved derivativestested for growth on minimal agar as compared to the input D(def=fmt)mutant. Stepwise increases in the growth rate of the evolving populationsuggest selection and fixation of successive adaptive mutations. We haveevidence that the protein met-tRNA synthetase, certain ribosomalproteins, initiation factor 2, and methionine aminopeptidase are alteredin the output strains.

[0046] The evolutionary process can be accelerated by increasingvariation in the population (FIG. 3). When mutation rates in thepopulation under selection were increased by a factor of about 1,000,wild-type growth rates were approximated within about half the timerequired for the process shown in FIG. 2a.

[0047] Current technology for continuous proliferation of cells insuspension suffers a major drawback, selection of adhesive variantswhich stick to inner surfaces of the device and escape the selectivepressure imposed by continuous or conditional dilution (Chao, L. andRamsdell, G. (1985): The effects of wall populations on coexistence ofbacteria in the liquid phase of chemostat cultures. J. Gen. Microbiol.131, 1229-1236). In principle, this can be avoided by serial subcultureof cells in suspension (Lenski, R. E. and, Travisano, M. (1994):Dynamics of adaptation and diversification: A 10,000-generationexperiment with bacterial populations. Proc. Natl. Acad. Sci. USA 91,6808-6814), a technique where cells in suspension are frequentlytransferred into fresh culture vessels (i.e., surfaces are periodicallydiscarded), creating a selective disadvantage for static variants. At anindustrial scale, serial subculture technology has not beensystematically exploited because it is laborious and requires absolutesterility during transfers.

[0048]FIG. 4 automated technology for the permanent proliferation ofpopulations of cells exclusively in suspension. During the course of anexperiment similar to that shown in FIG. 3, operation of the device wasmanipulated such that static, adhesive variants were no longer destroyedand could freely compete with cells in suspension. Highly adhesivevariants accumulated rapidly (FIG. 4b). In parallel, the growth rate ofthe population decreased, demonstrating that these static variants arenot subject to the selective pressure imposed on the cells insuspension. When proper operation of the device was re-established,these variants were rapidly and effectively eliminated from the evolvingpopulation.

[0049] Conclusion

[0050] 1) the automated device describe in PCT WO 00/34433 is the firstoperational apparatus which allows permanent proliferation of livingcells under defined, selective conditions.

[0051] 2) The automated process frequently and effectively destroysstatic variants in any part of the device, overcoming the primaryobstacle to continuous proliferation of cells in suspension forindefinite periods of time.

[0052] 3) We have created derivatives of Escherichia coli with a primermethionine cycle similar to that in eukaryotic cells. The strains willallow for expression of N-formyl-free polypeptides in E. coli.

[0053] 4) Evolved microbial strains with unique genetic and metabolicimprints will serve as ancestors for the diversification of lines ofindustrially fit microorganisms. Our reference strain is 2045 :Escherichia coli MG16555 (def-fmt):: cat, dnaQ::miniTn10 [CM^(R),Tc^(R)]

[0054] MG 1655 is a wild-type K12 strain of E. coli (see EMBO J. (1994)13:914-923) v(def-fmt) means deletion of the def-fmt operon, whichencodes the polypeptide deformylase and the Met-LRNAi transformylaseactivities. This allele has been described (EMBO J. (1994) 13:914-923).

[0055] The writing::cat means the insertion of a cat (chloramphenicolacetyl-transferase) germ in the (def-fmt) locus dnaQ::miniTn10 meansthat the dnaQ gene (epsilon subunit of the DNA polymerase, theproof-reading subunit) is interrupted by the insertion of aminitransposon Tn10 which confers the tetracycline resistance [CmR, TcR]means that the strain is resistant to chloramphenicol (25 micg/ml) andto Tetracycline (15 micg/ml).

[0056] This bacterial strain caries a deletion of the def-fmt operon andis consequently defective in the Met-tRNA1 transformylase andpolypeptide deformylase activities. This strain also carries dnaQmutation and consequently shows a mutator phenotype. This strain is aderivative of 2124 that has been selected to grow in minimal and complexnutrient media at 30° C., 37° C. and 42° C. with near wild-type rate(approximately 25 min in LB and approximately 80 min in MS minimalmedium (Richard (1993), J. Biol. Chem. 268:26827-26835) with mannitol ascarbon source at final concentration 0.2% at 37 C.). The original 2124strain shows a growth rate of approximately 200 min in MS minimalmedium+mannitol at 37° C. 2045, the 2124 derivative, was selected forenhanced growth rates under permanent proliferation in minimal medium at37° C. until its growth rate reached that of MG 1655, the parentwild-type bacterium.

[0057] The mutator phenotype can be rescued by complementation with adnaQ wild type allele expressed either from a plasmid or from thechromosome, through an allele replacement in 2045.

[0058] Subject: 2137

[0059] Our reference strain is 2137: Escherichia coli MG165, fmt::cat[44° CS, CmR] MG1655 is a wild-type K12 strain of E. coli (see EMBO J.(1994) 13:914-923). fmt means deletion of the fmt gene which encodes theMet-tRNAi transformylase activity. This allele has never been described,it is a Pst I deletion, internal to fmt (nucleotides 247 to 484).

[0060] The writing ::cat means the insertion of a cat (chloramphenicolacetyl-transferase) gene at the PstI site of the deletion.

[0061] The cat gene is identical to the one used for the construction ofthe (def-fmt)::cat allele (see EMBO J. (1994) 13:914-923). [44 CS, CmR]means that the strain is thermosensitive and resistant tochloramphenicol (25 micg/ml).

[0062] This bacterial strain carries a deletion of the fmt gene and isconsequently defective in the Met-tRNAi transformylase activity.

[0063] This strain is a derivative of MG1655 that has a growth rate ofapproximately 200 min in MS minimal medium+mannitol at 37° C.

1 4 1 169 PRT Escherichia coli 1 Met Ser Val Leu Gln Val Leu His Ile ProAsp Glu Arg Leu Arg Lys 1 5 10 15 Val Ala Lys Pro Val Glu Glu Val AsnAla Glu Ile Gln Arg Ile Val 20 25 30 Asp Asp Met Phe Glu Thr Met Tyr AlaGlu Glu Gly Ile Gly Leu Ala 35 40 45 Ala Thr Gln Val Asp Ile His Gln ArgIle Ile Val Ile Asp Val Ser 50 55 60 Glu Asn Arg Asp Glu Arg Leu Val LeuIle Asn Pro Glu Leu Leu Glu 65 70 75 80 Lys Ser Gly Glu Thr Gly Ile GluGlu Gly Cys Leu Ser Ile Pro Glu 85 90 95 Gln Arg Ala Leu Val Pro Arg AlaGlu Lys Val Lys Ile Arg Ala Leu 100 105 110 Asp Arg Asp Gly Lys Pro PheGlu Leu Glu Ala Asp Gly Leu Leu Ala 115 120 125 Ile Cys Ile Gln His GluMet Asp His Leu Val Gly Lys Leu Phe Met 130 135 140 Asp Tyr Leu Ser ProLeu Lys Gln Gln Arg Ile Arg Gln Lys Val Glu 145 150 155 160 Lys Leu AspArg Leu Lys Ala Arg Ala 165 2 510 DNA Escherichia coli 2 atgtcagttttgcaagtgtt acatattccg gacgagcggc ttcgcaaagt tgctaaaccg 60 gtagaagaagtgaatgcaga aattcagcgt atcgtcgatg atatgttcga gacgatgtac 120 gcagaagaaggtattggcct ggcggcaacc caggttgata tccatcaacg tatcattgtt 180 attgatgtttcggaaaaccg tgacgaacgg ctagtgttaa tcaatccaga gcttttagaa 240 aaaagcggcgaaacaggcat tgaagaaggt tgcctgtcga tccctgaaca acgtgcttta 300 gtgccgcgcgcagagaaagt taaaattcgc gcccttgacc gcgacggtaa accatttgaa 360 ctggaagcagacggtctgtt agccatctgt attcagcatg agatggatca cctggtcggc 420 aaactgtttatggattatct gtcaccgctg aaacaacaac gtattcgtca gaaagttgaa 480 aaactggatcgtctgaaagc ccgggcttaa 510 3 315 PRT Escherichia coli 3 Met Ser Glu SerLeu Arg Ile Ile Phe Ala Gly Thr Pro Asp Phe Ala 1 5 10 15 Ala Arg HisLeu Asp Ala Leu Leu Ser Ser Gly His Asn Val Val Gly 20 25 30 Val Phe ThrGln Pro Asp Arg Pro Ala Gly Arg Gly Lys Lys Leu Met 35 40 45 Pro Ser ProVal Lys Val Leu Ala Glu Glu Lys Gly Leu Pro Val Phe 50 55 60 Gln Pro ValSer Leu Arg Pro Gln Glu Asn Gln Gln Leu Val Ala Glu 65 70 75 80 Leu GlnAla Asp Val Met Val Val Val Ala Tyr Gly Leu Ile Leu Pro 85 90 95 Lys AlaVal Leu Glu Met Pro Arg Leu Gly Cys Ile Asn Val His Gly 100 105 110 SerLeu Leu Pro Arg Trp Arg Gly Ala Ala Pro Ile Gln Arg Ser Leu 115 120 125Trp Ala Gly Asp Ala Glu Thr Gly Val Thr Ile Met Gln Met Asp Val 130 135140 Gly Leu Asp Thr Gly Asp Met Leu Tyr Lys Leu Ser Cys Pro Ile Thr 145150 155 160 Ala Glu Asp Thr Ser Gly Thr Leu Tyr Asp Lys Leu Ala Glu LeuGly 165 170 175 Pro Gln Gly Leu Ile Thr Thr Leu Lys Gln Leu Ala Asp GlyThr Ala 180 185 190 Lys Pro Glu Val Gln Asp Glu Thr Leu Val Thr Tyr AlaGlu Lys Leu 195 200 205 Ser Lys Glu Glu Ala Arg Ile Asp Trp Ser Leu SerAla Ala Gln Leu 210 215 220 Glu Arg Cys Ile Arg Ala Phe Asn Pro Trp ProMet Ser Trp Leu Glu 225 230 235 240 Ile Glu Gly Gln Pro Val Lys Val TrpLys Ala Ser Val Ile Asp Thr 245 250 255 Ala Thr Asn Ala Ala Pro Gly ThrIle Leu Glu Ala Asn Lys Gln Gly 260 265 270 Ile Gln Val Ala Thr Gly AspGly Ile Leu Asn Leu Leu Ser Leu Gln 275 280 285 Pro Ala Gly Lys Lys AlaMet Ser Ala Gln Asp Leu Leu Asn Ser Arg 290 295 300 Arg Glu Trp Phe ValPro Gly Asn Arg Leu Val 305 310 315 4 948 DNA Escherichia coli 4gtgtcagaat cactacgtat tatttttgcg ggtacacctg actttgcagc gcgtcatctc 60gacgcgctgt tgtcttctgg tcataacgtc gttggcgtgt tcacccagcc agaccgaccg 120gcaggacgcg gtaaaaaact gatgcccagc ccggttaaag ttctggctga ggaaaaaggt 180ctgcccgttt ttcaacctgt ttccctgcgt ccacaagaaa accagcaact ggtcgccgaa 240ctgcaggctg atgttatggt cgtcgtcgcc tatggtttaa ttctgccgaa agcagtgctg 300gagatgccgc gtcttggctg tatcaacgtt catggttcac tgctgccacg ctggcgcggt 360gctgcaccaa tccaacgctc actatgggcg ggtgatgcag aaactggtgt gaccattatg 420caaatggatg tcggtttaga caccggtgat atgctctata agctctcctg cccgattact 480gcagaagata ccagtggtac gctgtacgac aagctggcag agcttggccc acaagggctt 540atcaccacgt tgaaacaact ggcagacggc acggcgaaac cagaagttca ggacgaaact 600cttgtcactt acgccgagaa gttgagtaaa gaagaagcgc gtattgactg gtcactttcg 660gcagcacagc ttgaacgctg cattcgcgct ttcaatccat ggccaatgag ctggctggaa 720attgaaggac agccggttaa agtctggaaa gcatcggtca ttgatacggc aaccaacgct 780gcaccaggaa cgatccttga agccaacaaa caaggcattc aggttgcgac tggtgatggc 840atcctgaacc tgctctcgtt acaacctgcg ggtaagaaag cgatgagcgc gcaagacctc 900ctgaactctc gtcgggaatg gtttgttccg ggcaaccgtc tggtctga 948

1: use of a bacterial strain lacking of deformylase and/or transformylase activities for the obtention of non formylated peptides or proteins. 2: use of a bacterial strain lacking of polypeptide deformylase and/or Met-tRNA transformylase activities for the obtention of non formylated peptides or proteins. 3: use of a bacterial strain according to claims 1 to 2 lacking of deformylase and/or transformylase activities characterized in that it is non reversible for the deformylase and/or transformylase activities 4: use of the bacterial strain according to claim 1 to 3 characterized in that it is cultivated at least at a temperature of 37° C. 5: use of a bacterial strain according to claim 1 and 4 characterized in that the bacteria is E. coli 6: A bacterial strain lacking of the deformylation and/or transformylation activity characterized in that said bacteria is not able to revert spontaneousely for these activities, and the growth rate of said bacteria is at least equivalent to the wild type growth rate. 7: use of the bacteria according to claim 6 for the production of non formylated peptides or proteins. 8: A process for producing peptides or proteins comprising the following steps: a) culture of the bacteria according to claim 6 b) transforming the bacteria according to step a) with a DNA molecule or a plasmid or a vector comprising an insert containing a polynucleotide coding for an heterologous or homologous peptide or protein c) production of the peptide or the protein by the bacteria d) optionaly, separation of the peptide or protein of interest from the bacterial culture. 9: a purified polynucleotide comprising a gene coding for a mutator allele and contained in the bacteria strain according to claim 6 10: a purified polynucleotide according to claim 9 in which the mutator phenotype has been complemented or the mutator allele has been replaced by the wild type allele 11: process according to claim 8 in which the bacteria comprises a polynucleotide coding for a mutator allele. 12: Process according to claim 8 in which the bacteria mutator phenotype has been complemented or the mutator allele has been replaced by the wid type allele 13: E. coli bacterial strain γ2045 culture deposit at the CNCM (accesion number=I-2694 on Jul. 5, 2001 ) 14: A process for the selection of mutants of a bacteria according to claim 6 characterized by the culture under continuous proliferation conditions of said mutants and separation of the mutant of interest from the static bacteria 15: Purified protein obtained after the expression of the heterologous insert of interest and deprived of formyl residue 